Cranial Neural crest cells (CNC) are an embryonic population of multipotent precursor cells that give rise to craniofacial cartilage and bone in the head. Understanding the mechanisms responsible for specification, migration and differentiation of the CNC involves characterizing multiple signaling pathways that converge to regulate expression of effectors genes. Recently, we have shown that Alk8, a novel Type 1 TGF-beta family member receptor, participates in Bmp signaling pathways and influences mediolateral positioning of CNC's and dorsoventral (DV) patterning of pharyngeal arch cartilages. The goal of this proposal is to define the role of Alk8 and Bmp signaling in pharyngeal arch morphogenesis and potential roles in specification of the neural crest. Zebrafish are in ideal vertebrate model for these studies because the embryos are transparent, easy to genetically manipulate, and the craniofacial skeleton is well formed within 5 days. To place Alk8 signals into the larger context of the multiple pathways regulating arch development, Specific Aim 1 is designed to screen for alk8 downstream signaling components. The results of this screen will provide a pool of potential candidates for CNC specific signaling events. The experiments outlined in Specific Aim 2 will characterize the temporospatial expression patterns of Alk8. These studies will be coupled with promoter analysis to determine regulatory elements driving tissue specific and/or temporally restricted Alk8 expression. Specific Aim 3, mosaic analysis, will determine the cell autonomous/non-autonomous effect that Alk8 experts on the neural crest. Specific Aim 4 will examine pharyngula stages of embryogenesis to determine if apoptosis or later chondrogenic defects contribute to defects in cartilage formation. These experiments will lend insight into the mechanisms specifying the neural crest, and will aid in development of therapies for congenital disorders such as Treacher Colins syndrome, Hirschsprung's disease and Cleidocranial dysplasia.